The present invention relates to noninvasive sampling of fetal cells for the purpose of prenatal diagnosis of genetic diseases. Candidate cells proposed by others include leukocytes, erythroid cells, and trophoblasts. Estimates of the amount of fetal blood which crosses into the maternal circulation in the first or second trimester include 2 .mu.L per day or a total of 50 to 200 .mu.L by the midtrimester (Parks et al.). Other estimates suggest that about 1/50,000 cells are fetal (Schroder et al. ). A recent study determined that this ratio increased from 1/144,000 at 15 weeks gestation to 1/4,000 later in pregnancy (Hamada et al.).
Abbreviations used herein include:
______________________________________ BFU-E Burst forming unit-erythroid BPA Burst promoting activities BSA Bovine serum albumin CFU-E Colony forming unit-erythroid Ep Erythropoietin FACS Fluorescence-activated cell sorting FCS Fetal calf serum FISH Fluorescence in situ hybridization GM-CSF Granulocyte-macrophage colony stimulating factor GPA Glycophorin A Hb Hemoglobin IL-3 Interleukin-3 IL-6 Interleukin-6 MACS Magnetic-activated cell sorting MNC Mononuclear cells MRNA Messenger ribonucleic acid PCR Polymerase chain reaction RT-PCR Reverse transcription PCR SCF Stem cell factor TFR Transferrin receptor TSPR Thrombospondin receptor ______________________________________
Zipursky et al. were the first to demonstrate fetal red cells in maternal blood (albeit, immediately following delivery), using the Kleihauer-Betke stain for fetal hemoglobin (Zipursky et al.). Schr oder reviewed this topic in 1975 and suggested that more than 1 fetal cell per 50,000 maternal cells was present in 5-10% of pregnancies in the second trimester (Schr oSder et al.). Parks and Herzenberg used fluorescence activated cell sorting (FACS) and found Rh D.sup.+ red cells in the blood of all Rh D.sup.- ABO compatible mothers in frequencies ranging from 1:4,000 to 1:80,000, corresponding to 200 .mu.L of fetal blood (Parks et al.). Fetal erythroblasts (and reticulocytes) in maternal blood were identified as early as 16 weeks gestation using fluorescence activated cell sorting (FACS) with monoclonal antibodies to the transferrin receptor (TFR) by Bianchi et al. (1990) Price et al. enhanced the enrichment with the addition of antibody to glycophorin A (GPA), and sorting according to cell size (forward angle light scatter) and granularity (side scatter). They concluded that there was an enrichment from 1 fetal nucleated red cell per 10.sup.7 maternal cells to 1 per 10-20 cells, using in situ hybridization with markers for X and Y chromosomes. G anshirt-Ahlert et al. suggested that magnetic-activated cell sorting (MACS) with microbeads would be faster than FACS, but found that antibody to TFR was not efficient for the enrichment of nucleated fetal cells, since many erythroblasts were non-reactive, and many reticulocytes (fetal and maternal) were TFR-positive. Bianchi et al. (1993) then combined antibodies to TFR, GPA, and to the thrombospondin receptor (TSPR), and found that GPA was the most important marker for recovery of fetal nucleated cells. Many of the analyses using TFR and GPA did not determine the final proportion of fetal/maternal cells; they used either polymerase chain reaction (PCR) amplification of DNA for Southern blots or fluorescence in situ hybridization (FISH) to detect male cells or fetal aneuploidy.
The presence of fetal white cells in the pregnant mother's blood was suggested as early as 1969 by Walknowska et al., based on the detection of XY karyotype in 0.2 to 1% of lymphocytes. In similar studies, examining for Y chromosomes or Y fluorescence in interphase cells, fetal lymphocytes comprised about 0.1 to 1% (Schroder et al., 1972; De Grouchy et al.; Schroder et al., 1975; Grosset, et al.; Siebers et al.; and Kirsch-Voiders et al.). Herzenberg et al. used FACS to sort for paternal HLA types, and found Y-positive cells at an incidence of 3 per 1,000 sorted cells; a larger study by the same group found an incidence of 1/800 to 1/60,000 (Iverson et al.). One group stained maternal mononuclear cells (MNC) for .alpha.-fetoprotein and found 1/1,000 positive cells (Kulozik et al.). Nakagome et al. used PCR amplification of Y DNA from unseparated maternal cells and found no positives, indicating that fetal cells were less than 1/25,000 maternal cells. However, Kao et al. were able to correctly identify male fetuses with this approach and indicated that 2 fetal cells were sufficient. Lo et al. used nested PCR with 2 sets of Y-specific primers, and could find 1 male in 300,000 female cells. To circumvent the possibility of residual lymphocytes from earlier male pregnancies, Wessman et al. used anti-My7 to identify granulocytes on cytospin slides of maternal mononuclear cells and found that about 0.1% were Y.sup.+ using in situ hybridization. Thus, the sensitivity of detection of fetal leukocytes in maternal blood varies widely, perhaps dependent on the assay method, the method of enrichment, and the gestational age; the specificity also varies, with most reports including false negatives as well as false positives.
Another cell type which was sought is trophoblasts, which might be distinguished with the monoclonal antibody H315 (Covone et al., 1984, and Mueller et al.). However, H315-positive cells were found in nonpregnant women (Pool et al. ), and the consensus is that nontrophoblast cells may absorb this antigen (Covone et al. 1988). Indeed, Bruch et al. reported that positive cells sorted with 3 monoclonal antibodies to trophoblasts had the morphology of leukocytes. Cacheux et al. depleted maternal lymphocytes with monoclonal antibodies and magnetic beads, and then used trophoblast antibodies and FACS; 4% of 1,000 recovered cells were Y-positive by FISH.
The major methods for cell separation have included labelling with monoclonal antibodies, and separating with FACS, with Dynal magnetic beads, or micromagnetic beads (MACS). After separation, the resulting semipurified cells are then examined using PCR amplification or in situ hybridization of specific genes or chromosomes or by standard karyotyping of metaphases.